Substituted 4,4&#39;-biphenylylene bis(2-thioureylene)di-naphthalenetrisulfonic acids and salts thereof

ABSTRACT

4,4&#39;-biphenylylene bis(2-thioureylene)di-naphthalenetrisulfonic acids and their salts which are useful as inhibitors of the complement system of warm-blooded animals, and the 8-isothiocyanato-1,3,6-naphthalenetrisulfonic acids and their salts which are new intermediates for the preparation of the active compounds.

DESCRIPTION OF THE INVENTION

This invention is concerned with pharmaceutically acceptable compoundshaving complement inhibiting activity which are of the formula (I):##STR1## wherein A is selected from the group consisting of alkali metaland alkaline earth metal; R is selected from the group consisting ofhydroxy and --SO₃ A, wherein A is as previously defined; and thepharmaceutically acceptable salts thereof.

A preferred embodiment of the instant invention consists of thosecompounds wherein A is as previously defined; and R is hydroxy. A secondpreferred embodiment consists of those compounds wherein A is aspreviously defined; and R is selected from the group consisting of --SO₃A, wherein A is as previously defined.

This invention is also concerned with compounds of formula II: ##STR2##wherein R and A are as previously defined, said compounds being usefulas intermediates for the preparation of the complement inhibitingcompounds of formula I.

The compounds of the present invention may be prepared according to thefollowing Flow Chart (A). ##STR3## wherein R and A are as hereinbeforedescribed.

The novel 8-isothiocyanato-1,3,6-naphthalenetrisulfonic acids and salts(II), which are useful intermediates, are prepared by converting the8-amino derivative to the 8-isothiocyanato derivative in the presence ofthiophosgene and aqueous HCl.

These intermediates (II) are then converted to the final products (I) byreaction with the appropriate 4,4'-diamino-2,2'-biphenyl disulfonic acidor salt (III) thereof with heat and finally with precipitation fromaqueous solution with ethanol.

The term "complement" refers to a complex group of proteins in bodyfluids that, working together with antibodies or other factors, play animportant role as mediators of immune, allergic, immunochemical and/orimmunopathological reactions. The reactions in which complementparticipates take place in blood serum or in other body fluids, andhence are considered to be humoral reactions.

With regard to human blood, there are at present more than 11 proteinsin the complement system. These complement proteins are designated bythe letter C and by number: C1, C2, C3 and so on up to C9. Thecomplement protein C1 is actually an assembly of subunits designatedC1q, C1r and C1s. The numbers assigned to the complement proteinsreflect the sequence in which they become active, with the exception ofcomplement protein C4, which reacts after C1 and before C2. Thenumerical assignments for the proteins in the complement system weremade before the reaction sequence was fully understood. A more detaileddiscussion of the complement system and its role in body processes canbe found in, for example, Bull. World Health Org., 39, 935-938 (1968);Ann. Rev. Medicine, 19, 1-24 (1968); The John Hopkins Med. J., 128,57-74 (1971); Harvey Lectures, 66, 75-104 (1972); The New EnglandJournal of Medicine, 287, 452-454; 489-495; 545-549; 592-596; 642-646(1972); Scientific American, 229, (No. 5), 54-66 (1973); FederationProceedings, 32, 134-137 (1973); Medical World News, Oct. 11, 1974, pp.53-58; 64-66; J. Allergy Clin. Immunol., 53, 298-302 (1974); Cold SpringHarbor Conf. Cell Proliferation 2/Proteases Biol. Control/229-241(1975); Annals of Internal Medicine, 84, 580-593 (1976); "Complement:Mechanisms and Functions", Prentice-Hall, Englewood Cliffs, N.J. (1976).

The complement system can be considered to consist of three sub-systems:(1) a recognition unit (C1q) which enables it to combine with antibodymolecules that have detected a foreign invader; (2) an activation unit(C1r, C1s, C2, C4, C3) which prepares a site on the neighboringmembrane; and (3) and attack unit (C5, C6, C7, C8 and C9) which createsa "hole" in the membrane. The membrane attack unit is non-specific; itdestroys invaders only because it is generated in their neighborhood. Inorder to minimize damage to the host's own cells, its activity must belimited in time. This limitation is accomplished partly by thespontaneous decay of activated complement and partly by interference byinhibitors and destructive enzymes. The control of complement, however,is not perfect, and there are times when damage is done to the host'scells. Immunity is therefore a double-edged sword.

Activation of the complement system also accelerates blood clotting.This action comes about by way of the complement-mediated release of aclotting factor from platelets. The biologically active complementfragments and complexes can become involved in reactions that damage thehost's cells, and these pathogenic reactions can result in thedevelopment of immune-complex diseases. For example, in some forms ofnephritis, complement damages the basal membrane of the kidney,resulting in the escape of protein from the blood into the urine. Thedisease disseminated lupus erythematosus belongs in this category; itssymptoms include nephritis, visceral lesions and skin eruptions. Thetreatment of diphtheria or tetanus with the injection of large amountsof antitoxin sometimes results in serum sickness, an immune-complexdisease. Rheumatoid arthritis also involves immune complexes. Likedisseminated lupus erythematosus, it is an autoimmune disease in whichthe disease symptoms are caused by pathological effects of the immunesystem in the host's tissues. In summary, the complement system has beenshown to be involved with inflammation, coagulation, fibrinolysis,antibody-antigen reactions and other metabolic processes.

In the presence of antibody-antigen complexes the complement proteinsare involved in a series of reactions which may lead to irreversiblemembrane damage if they occur in the vicinity of biological membranes.Thus, while complement constitutes a part of the body's defensemechanism against infection it also results in inflammation and tissuedamage in the immunopathological process. The nature of certain of thecomplement proteins, suggestions regarding the mode of complementbinding to biological membranes and the manner in which complementeffects membrane damage are discussed in Annual Review in Biochemistry,38, 389 (1969).

A variety of substances have been disclosed as inhibiting the complementsystem, i.e., as complement inhibitors. For example, the compounds3,3'-ureylenebis-[6-(2-amino-8-hydroxy-6-sulfo-1-naphthylazo)]benzenesulfonicacid, tetrasodium salt (chlorazol fast pink), heparin and a sulphateddextran have been reported to have an anticomplementary effect, BritishJournal of Experimental Pathology, 33, 327-339 (1952). The compound8-(3-benzamido-4-methylbenzamido)naphthalene-1,-3,5-trisulfonic acid(Suramin) is described as a competitive inhibitor of the complementsystem, Clin. Exp. Immunol., 10, 127-138 (1972). German Pat. No.2,254,893 or South African Pat. No. 727,923 discloses certain1-(diphenylmethyl)-4-(3-phenylallyl) piperazines useful as complementinhibitors. Other chemical compounds having complement inhibitingactivity are disclosed in, for example, Journal of Medicinal Chemistry,12, 415-419; 902-905; 1049-1052; 1053-1056 (1969); Canadian Journal ofBiochemistry, 47, 547-552 (1969); The Journal of Immunology, 93, 629-640(1964); The Journal of Immunology, 104, 279-288 (1970); The Journal ofImmunology, 106, 241-245 (1971); and The Journal of Immunology, 111,1061-1066 (1973).

It has been reported that the known complement inhibitorsepsilon-aminocapronic acid, Suramin and tranexamic acid all have beenused with success in the treatment of hereditary angioneurotic edema, adisease state resulting from an inherited deficiency or lack of functionof the serum inhibitor of the activated first component of complement(C1 inhibitor), The New England Journal of Medicine, 286, 808-812(1972).

The compounds of the present invention may be administered internally,e.g., orally, intra-articularly or parenterally, e.g., intra-articular,to a warm-blooded animal to inhibit complement in the body fluid of theanimal, such inhibition being useful in the amelioration or preventionof those reactions dependent upon the function of complement, such asinflammatory process and cell membrane damage induced byantigen-antibody complexes. A range of doses may be employed dependingon the mode of administration, the condition being treated and theparticular compound being used. For example, for intravenous orsubcutaneous use from about 5 to about 50 mg/kg/day, or every six hoursfor more rapidly excreted salts, may be used. For intra-articular usefor large joints such as the knee, from about 2 to about 20 mg/joint perweek may be used, with proportionally smaller doses for smaller joints.The dosage range is to be adjusted to provide optimum therapeuticresponse in the warm-blooded animal being treated. In general, theamount of compound administered can vary over a wide range to providefrom about 5 mg/kg to about 100 mg/kg of body weight of animal per day.The usual daily dosage for a 70 kg subject may vary from about 350 mg toabout 3.5 g. Unit doses of the acid or salt can contain from about 0.5mg to about 500 mg.

While in general the sodium salts of the acids of the invention aresuitable for parenteral use, other salts may also be prepared, such asthose of primary amines, e.g., ethylamine; secondary amines, e.g.,diethylamine or diethanol amine; tertiary amines, e.g., pyridine ortriethylamine or 2-dimethylaminomethyl-dibenzofuran; aliphatic diamines,e.g., decamethylenediamine; and aromatic diamines, can be prepared. Someof these are soluble in water, others are soluble in saline solution,and still others are insoluble and can be used for purposes of preparingsuspensions for injection. Furthermore as well as the sodium salt, thoseof the alkali metals, such as potassium and lithium; of ammonia; and ofthe alkaline earth metals, such as calcium or magnesium, may beemployed. It will be apparent, therefore, that these salts embrace, ingeneral derivatives of salt-forming cations.

In therapeutic use, the compounds of this invention may be administeredin the form of conventional pharmaceutical compositions. Suchcompositions may be formulated so as to be suitable for oral orparenteral administration. The active ingredient may be combined inadmixture with a pharmaceutically acceptable carrier, which carrier maytake a wide variety of forms depending on the form of preparationdesired for administration, i.e., oral or parenteral. The compounds canbe used in compositions such as tablets. Here, the principal activeingredient is mixed with conventional tabletting ingredients such ascorn starch, lactose, sucrose, sorbitol, talc, stearic acid, magnesiumstearate, dicalcium phosphate, gums, or similar materials as non-toxicpharmaceutically acceptable diluents or carriers. The tablets or pillsof the novel compositions can be laminated or otherwise compounded toprovide a dosage form affording the advantage of prolonged or delayedaction or predetermined successive action of the enclosed medication.For example, the tablet or pill can comprise an inner dosage and anouter dosage component, the latter being in the form of an envelope overthe former. The two components can be separated by an enteric layerwhich serves to resist disintegration in the stomach and permits theinner component to pass intact into the duodenum or to be delayed inrelease. A variety of materials can be used for such enteric layers orcoatings, such materials including a number of polymeric acids ormixtures of polymeric acids with such materials as shellac, shellac andcetyl alcohol, cellulose acetate and the like. A particularlyadvantageous enteric coating comprises a styrene maleic acid copolymertogether with known materials contributing to the enteric properties ofthe coating. The tablet or pill may be colored through the use of anappropriate non-toxic dye, so as to provide a pleasing appearance.

The liquid forms in which the novel compositions of the presentinvention may be incorporated for administration include suitableflavored emulsions with edible oils, such as, cottonseed oil, sesameoil, coconut oil, peanut oil, and the like, as well as elixirs andsimilar pharmaceutical vehicles. Sterile suspensions or solutions can beprepared for parenteral use. Isotonic preparations containing suitablepreservatives are also desirable for injection use.

The term dosage form, as described herein, refers to physically discreteunits suitable as unitary dosage for warm-blooded animal subjects, eachunit containing a predetermined quantity of active component calculatedto produce the desired therapeutic effect in association with therequired pharmaceutical diluent, carrier or vehicle. The specificationfor the novel dosage forms of this invention are indicated bycharacteristics of the active component and the particular therapeuticeffect to be achieved or the limitations inherent in the art ofcompounding such an active component for therapeutic use in warm-bloodedanimals as disclosed in this specification. Examples of suitable oraldosage forms in accord with this invention are tablets, capsules, pills,powder packets, granules, wafers, cachets, teaspoonfuls, dropperfuls,ampules, vials, segregated multiples of any of the foregoing and otherforms as herein described.

The complement inhibiting activity of the compounds of this inventionhas been demonstrated by one or more of the following identified tests:(i) Test, Code 026 (C1 inhibitor) -- This test measures the ability ofactivated human C1 to destroy fluid phase human C2 in the presence of C4and appropriate dilutions of the test compound. An active inhibitorprotects C2 from C1 and C4; (ii) Test, Code 035 (C3-C9 inhibitor) --This test determines the ability of the late components of humancomplement (C3-C9) to lyse EAC 142 in the presence of appropriatedilutions of the test compound. An active inhibitor protects EAC 142from lysis by C3-C9; (iii) Test, Code 036 (C-Shunt inhibitor) -- In thistest human erythrocytes rendered fragile are lysed in autologous serumvia the shunt pathway activated by cobra venom factor in the presence ofappropriate dilutions of the test compound. Inhibition of the shuntpathway results in failure of lysis; (iv) Forssman Vasculitis Test --Here, the well known complement dependent lesion, Forssman vasculitis,is produced in guinea pigs by intradermal injection of rabbitanti-Forssman antiserum. The lesion is measured in terms of diameter,edema and hemorrhage and the extent to which a combined index of theseis inhibited by prior intraperitoneal injection of the test compound at200 mg/kg is then reported, unless otherwise stated; (v) Forssman ShockTest -- Lethal shock is produced in guinea pigs by an i.v. injection ofanti-Forssman antiserum and the harmonic mean death time of treatedguinea pigs is compared with that of simultaneous controls; (vi)Complement Level Reduction Test -- In this test, the above dosed guineapigs, or others, are bled for serum and the complement level isdetermined in undiluted serum by the capillary tube method of U.S. Pat.No. 3,876,376 and compared to undosed control guinea pigs; and (vii) Cap50 Test -- Here, appropriate amounts of the test compound are added to apool of guinea pig serum in vitro, after which the undiluted serumcapillary tube assay referred to above is run. The concentration ofcompound inhibiting 50% is reported.

With reference to Table I, guinea pigs weighing about 300 g were dosedintravenously (i.v.) or intraperitoneally (i.p.) with 200 mg/kg of thetest compound dissolved in saline and adjusted to pH 7-8. One hour afterdosing, the guinea pigs were decapitated, blood was collected and theserum separated. The serum was tested for whole complement using thecapillary tube assay. Percent inhibition was calculated by comparisonwith simultaneous controls. The results appear in Table I together withresults of tests, code 026, 035, 036, Cap 50, % inhibition and Forssmanshock. Table I shows that the compounds of the invention possess highlysignificant in vitro and in vivo, complement inhibiting activity inwarm-blooded animals.

                                      TABLE I                                     __________________________________________________________________________    Biological Activities                                                                                            In Vivo Activity (Guinea Pig)                                                 % Inhibition                                               Cl  C-Late                                                                            Shunt Inhi-                                                                              Intraperitoneal                                                                        Intravenous                                       026*                                                                              035*                                                                              bition 036*                                                                              Time(min.)                                                                             Time(min.)                        Compound        Wells                                                                             Wells                                                                             Wells Cap 50*                                                                            30 60 120                                                                              2  30 120                         __________________________________________________________________________    8,8'-[2,2'-Disulfo-4,4'-biphenylyl-                                           ene)bis(2-thioureylene)]-di-1,3,6-                                                            + 8 N   N     125  -55                                                                              -55                                                                              -70                                                                              -92                                                                              -46                                                                              -31                         naphthalenetrisulfonic acid octa-                                             sodium salt                                                                   __________________________________________________________________________     *Code designation for tests employed as referred herein.                      **Activity in wells a serial dilution assay. Higher well number indicates     higher activity. The serial dilutions are two-fold.                           N = Negative                                                             

EXAMPLE 1 8-Isothiocyanato-1,3,6-naphthalenetrisulfonic acid trisodiumsalt

To a solution of 78.0 g of 8-amino-1,3,6-naphthalenetrisulfonic acid,disodium salt in 600 ml of water containing 15 ml of concentratedhydrochloric acid at 25° C is added 25 g of thiophosgene and the mixtureis sitrred vigorously at 15°-20° C (water cooling bath) for 3 hours. Thesolution is evacuated at the water pump to remove residual thiophosgeneand then is treated with charcoal and filtered through diatomaceousearth. The filtrate is neutralized, portionwise, with sodium bicarbonateand is evaporated in vacuo to approximately 250 ml. The resultingconcentrated solution is allowed to crystallize overnight at 5° C. Thethick mixture is filtered and the product is washed with a small portionof ice-water, and then with acetone followed by diethyl ether. On dryingovernight in the Abderhalden at 110° C, there is obtained 63.3 g of thedesired isothiocyanate as a pale yellow powder.

EXAMPLE 2 4,4'-Diamino-2,2'-biphenyldisulfonic acid disodium salt

A solution of 31.4 g of 4,4'-diamino-2,2'-biphenyldisulfonic acid in 150ml of water containing 7.7 g of sodium hydroxide is filtered and dilutedwith 300 ml of ethanol. The mixture is filtered and the solid is washedwith ethanol and ether and dried overnight in vacuo at 110° C giving31.9 g of the desired final product.

EXAMPLE 38,8'-[(2,2'-Disulfo-4,4'-biphenylylene)bis(2-thioureylene)]di-1,3,6-naphthalenetrisulfonicacid octasodium salt

A solution of 0.98 g of 4,4'-diamino-2,2'-biphenyldisulfonic aciddisodium salt in 5 ml of water is prepared. To this is added a warmsolution of 2.55 g of 8-isothiocyanato-1,3,6-naphthalenetrisulfonic acidtrisodium salt in 7 ml of water. The mixture is allowed to stand at roomtemperature for 15 minutes, heated on a steam bath for 5 minutes,treated with charcoal, filtered through diatomaceous earth andconcentrated in vacuo to about 5 ml. This concentrate is diluted with 75ml of ethanol, stirred until solid and then ground in a mortar. Thesolid is washed with ethanol and ether and dried overnight in vacuo at110° C giving 3.43 g of the desired final product as a pale yellowsolid.

EXAMPLE 4

    ______________________________________                                        Preparation of Compressed Tablet                                              Ingredient              mg/Tablet                                             ______________________________________                                        Active Compound          0.5-500                                              Dibasic Calcium Phosphate N.F.                                                                        qs                                                    Starch USP              40                                                    Modified Starch         10                                                    Magnesium Stearate USP  1-5                                                   ______________________________________                                    

EXAMPLE 5

    ______________________________________                                        Preparation of Compressed Tablet - Sustained Action                           Ingredient            mg/Tablet                                               ______________________________________                                        Active Compound       0.5-500(as acid                                         as Aluminum Lake*, Micronized                                                                       equivalent)                                             Dibasic Calcium Phosphate N.F.                                                                      qs                                                      Alginic Acid          20                                                      Starch USP            35                                                      Magnesium Stearate USP                                                                              1-10                                                    ______________________________________                                         *Complement inhibitor plus aluminum sulfate yields aluminum complement        inhibitor. Complement inhibitor content in aluminum lake ranges from          5-30%.                                                                   

EXAMPLE 6

    ______________________________________                                        Preparation of Hard Shell Capsule                                             Ingredient            mg/Capsule                                              ______________________________________                                        Active Compound       0.5-500                                                 Lactose, Spray Dried  qs                                                      Magnesium Stearate    1-10                                                    ______________________________________                                    

EXAMPLE 7

    ______________________________________                                        Preparation of Oral Liquid (Syrup)                                            Ingredient            % W/V                                                   ______________________________________                                        Active Compound       0.05-5                                                  Liquid Sugar          75.0                                                    Methyl Paraben USP    0.18                                                    Propyl Paraben USP    0.02                                                    Flavoring Agent       qs                                                      Purified Water qs ad  100.0                                                   ______________________________________                                    

EXAMPLE 8

    ______________________________________                                        Preparation of Oral Liquid (Elixir)                                           Ingredient            % W/V                                                   ______________________________________                                        Active Compound       0.05-5                                                  Alcohol USP           12.5                                                    Glycerin USP          45.0                                                    Syrup USP             20.0                                                    Flavoring Agent       qs                                                      Purified Water qs ad  100.0                                                   ______________________________________                                    

EXAMPLE 9

    ______________________________________                                        Preparation of Oral Suspension (Syrup)                                        Ingredient            % W/V                                                   ______________________________________                                        Active Compound       0.05-5                                                  as Aluminum Lake, Micronized                                                                        (acid equivalent)                                       Polysorbate 80 USP    0.1                                                     Magnesium Aluminum Silicate                                                   Colloidal             0.3                                                     Flavoring Agent       qs                                                      Methyl Paraben USP    0.18                                                    Propyl Paraben USP    0.02                                                    Liquid Sugar          75.0                                                    Purified Water qs ad  100.0                                                   ______________________________________                                    

EXAMPLE 10

    ______________________________________                                        Preparation of Injectable Solution                                            Ingredient             % W/V                                                  ______________________________________                                        Active Compound        0.05-5                                                 Benzyl Alcohol N.F.    0.9                                                    Water for Injection    100.0                                                  ______________________________________                                    

EXAMPLE 11

    ______________________________________                                        Preparation of Injectable Oil                                                 Ingredient             % W/V                                                  ______________________________________                                        Active Compound        0.05-5                                                 Benzyl Alcohol 1.5                                                            Sesame Oil qs ad       100.0                                                  ______________________________________                                    

EXAMPLE 12

    ______________________________________                                        Preparation of Intra-Articular Product                                        Ingredient               Amount                                               ______________________________________                                        Active Compound          2-20 mg                                              NaCl (physiological saline)                                                                            0.9%                                                 Benzyl Alcohol           0.9%                                                 Sodium Carboxymethylcellulose                                                                          1-5%                                                 pH adjusted to 5.0-7.5                                                        Water for Injection qs ad                                                                              100%                                                 ______________________________________                                    

EXAMPLE 13

    ______________________________________                                        Preparation of Injectable Depo Suspension                                     Ingredient            % W/V                                                   ______________________________________                                        Active Compound       0.05-5                                                                        (acid equivalent)                                       Polysorbate 80 USP    0.2                                                     Polyethylene Glycol 4000 USP                                                                        3.0                                                     Sodium Chloride USP   0.8                                                     Benzyl Alcohol N.F.   0.9                                                     HCl to pH 6-8         qs                                                      Water for Injection qs ad                                                                           100.0                                                   ______________________________________                                    

We claim:
 1. A compound of the formula: ##STR4## wherein R is selectedfrom the group consisting of hydroxy and --SO₃ A, and A is apharmaceutically acceptable salt cation.
 2. A compound according toclaim 1, wherein R is hydroxy.
 3. A compound according to claim 1,wherein R is --SO₃ A.
 4. The compound according to claim 3,8,8'-[(2,2'-disulfo-4,4'-biphenylylene)bis(2-thioureylene)]di-1,3,6-naphthalenetrisulfonicacid octasodium salt.